Proteins in type 2 diabetes

ABSTRACT

The present invention relates to the use of naturally occurring compounds and derivatives thereof as markers for predisposition of diabetes related diseases. The invention also relates to a pharmaceutical composition for treatment of the diabetes related diseases.

FIELD OF INVENTION

The present invention relates to a method for diagnosis or prediction ofpredisposition of diabetes related diseases and pharmaceuticalcompositions for treatment of diabetes-related diseases.

BACKGROUND OF THE INVENTION

Diabetes is sub-divided on clinical grounds into insulin-dependent andnon-insulin dependent diabetes mellitus (IDDM (type 1) and NIDDM (type2) respectively). The two forms of the disease are distinguished by anumber of features.

In type 1 diabetes there is profound insulin deficiency such that eventhe low levels of insulin which would normally prevent lipolysis andcytogenesis cannot be sustained. Type 1 patients therefore generallyshow high levels of glucose and low levels of insulin. As the diseaseprogresses, the pancreatic islets are damaged or destroyed, and less andless insulin can be produced.

Type 2 diabetes is a common and complex disorder, which results from acombination of defects in insulin secretion and impaired insulinsensitivity (insulin resistance) in peripheral tissues, e.g. in skeletalmuscle (1). Type 2 diabetes is characterized by hyperglycaemia in boththe fasted and fed states, variable degrees of hyperinsulinaemia andobesity. Despite intensive investigation of proteins in insulinsignaling pathways in the past decade, the primary cellular causeremains uncertain. Recent reports of reduced oxidative enzyme activityin type 2 diabetic muscle (2), and of mitochondrial DNA mutationscausing type 2 diabetes through impairment of oxidative phosphorylation(3,4), add to the molecular complexity of this disease.

Current therapy includes diet, sulphonylurea to enhance insulinsecretion, insulin itself, and biguanides to reduce insulin resistance.There is a need for new antidiabetic agents, since biguanides are quitetoxic while sulphonylurea is ineffective in patients with severelyimpaired islet cell function, and after 10 years of treatment, 50% ofpatients will have become resistant.

A number of strategies have been employed in order to determining thepredisposition of diabetes. In WO 00/66762, to which reference is made,it is described that specific mutations in the mitochondrial gene ATPsynthase 8/6 sequence can be related to diabetes.

Further, WO 00/66782 and WO 98/17826 describe a method for diagnosingdiabetes by finding mutations in an ATP synthase gene.

Though several strategies for determining the risk of developingdiabetes have been suggested, no strategy has proven successful. Thepresent invention therefore fulfils the long-felt need for a method fordetermining the risk of developing diabetes.

SUMMARY OF THE INVENTION

The present inventors have invented a method for diagnosing anddetermining the predisposition of at least one disease relating todiabetes by measuring the level of a protein in a sample. The inventionis based on the finding that fifteen proteins surprisingly were eitherdown- or up-regulated in type 2-diabetes. Eleven of these proteins(markers for type 2 diabetes) were positively identified by massspectrometry. The observed changes in the expression of these elevenproteins are consistent with increased cellular stress and perturbationsin skeletal muscle mitochondrial metabolism in insulin resistantsubjects (2). Most of the proteins identified were eitherphosphoproteins or proteins involved in phosphate metabolism in thecell. Most interestingly, the present inventors demonstrated that ATPsynthase beta-subunit is phosphorylated, and found the expression of abeta-subunit phospho-isoform to be reduced and to correlate inverselywith fasting plasma glucose levels in diabetic muscle. These dataindicate a role for phosphorylation of ATP synthase beta-subunit in theregulation of ATP synthesis, and that alterations in the regulation ofthis protein and cellular stress proteins may contribute to pathogenesisof type 2 diabetes.

With regard to a method of treating diabetes, a single protein may betargeted for therapy or a grouping of proteins may be targeted. Thelevel of expression of these targeted proteins may be altered (e.g.increased or decreased) or the proteins themselves may be interferedwith in a method of preventing or delaying the onset of a diabetesrelated disease in a human according to the invention. This may beaccomplished by administering e.g. a marker protein, a nucleotidesequence (or complementary sequence or part thereof) coding for a markerprotein, an antibody for a marker protein, a nucleic acid fragmentcapable of binding to a marker protein, and/or a compound capable ofbinding to a marker protein to said human. Compounds which affect thetranscription of a marker or effect the post-translational modificationof a marker, and especially compounds which affect the activity(phosphorylation status) of a marker protein, would also be obvious drugcandidates.

DETAILED DISCLOSURE OF THE INVENTION

The present invention relates to a substantially pure polypeptide, whichcomprises at least one amino acid sequence selected from the groupconsisting of:

-   -   (a) ATP synthase beta subunit or phosphorylated ATP synthase        beta subunit isoforms;    -   (b) phosphoglucomutase 1;    -   (c) heat shock protein 90 beta;    -   (d) creatine kinase B subunit;    -   (e) myosin regulatory light chain 2;    -   (f) collagen alpha1 (VI) chain;    -   (g) 78 kDa glucose-regulated protein;    -   (h) a marker protein defined by the characteristics disclosed in        table 3 (match nos: 199, 303, 391, or 511);    -   (i) an analogue having preferably at least 70% (such as at least        80%, at least 90%, at least 95%) homology with any one of the        polypeptides in (a), (b), (c), (d), (e), (f), (g) or (h) or a        part thereof; and    -   (j) a derivative, precursor, analogue or modification of an        amino acid sequence in (a), (b), (c), (d), (e), (f), (g), (h) or        (i).

ATP synthase beta subunit is a nuclear encoded protein, and the subunitis placed in the catalytic part of the ATP synthase enzyme (in the F1part), in contrast to the ATP synthase 6/8 subunits which aremitochondial encoded proteins, and are placed in the catalytic inactivemembrane spanning part of the ATP synthase enzyme (in the F0 part).

The polypeptides, including their phosphorylated isoforms, can be usedas a medication, e.g. for treatment of at least one diabetes-relateddisease, preferably type 2 diabetes.

The invention further relates to a pharmaceutical composition fortreatment of at least one diabetes-related disease, preferably type 2diabetes, which comprises at least one polypeptide according to theinvention or a mimetic or an antimimetic thereof. Other pharmaceuticalcompositions according to the invention are such compositions whichcomprise:

-   -   (a) at least one substance which is capable of regulating the        expression of a nucleic acid fragment coding for at least a part        of a polypeptide according to the invention; and/or    -   (b) at least one said polypeptide; and/or    -   (c) at least one antibody raised against said polypeptide or its        modification product; and/or    -   (d) at least one nucleic acid fragment capable of hybridizing to        a nucleotide sequence including its regulatory elements encoding        said polypeptide or to the complementary strand; and/or    -   (e) at least one compound, e.g. a nucleic acid fragment, capable        of binding to said polypeptide; and/or    -   (f) at least one compound capable of modifying the protein form        associated with the disease so that its relative expression,        activity and/or concentration is changed towards that seen in        healthy persons; and/or    -   (g) at least one compound capable of marking (e.g. with        ubiquinone) at least one of said proteins which protein is        expressed at too high relative levels in the disease for more        rapid turnover or degradation; and/or    -   (h) at least one compound capable of marking at least one of        said proteins which protein is expressed at too low relative        levels for slower turnover or degradation; and/or    -   (i) an aptamer which binds to said protein or the site at which        said protein binds; and/or    -   (j) an aptamer which binds preferably to either the binding site        or the active site of said protein.

The pharmaceutical composition comprises at least one active compound,e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different compounds.

In another embodiment, the invention relates to a method of treating adiabetes-related disease, preventing said disease and/or delaying theonset of said disease in a human, which method comprising administeringat least one compound chosen from the group consisting of:

-   -   (a) a polypeptide according to the invention;    -   (b) a nucleotide sequence coding for the polypeptide in (a);    -   (c) an antibody able to bind the polypeptide in (a);    -   (d) a nucleic acid fragment capable of hybridizing to a nucleic        acid encoding at least a part of the polypeptide in (a) or the        complementary strand to said nucleic acid, including its        regulatory elements;    -   (e) a compound capable of binding to the polypeptide in (a);    -   (f) a compound capable of up- or down regulating the expression,        activity and/or concentration of at least one polypeptide        according to the invention;    -   (g) a compound capable of modifying the protein form associated        with the disease so that its relative expression, activity        and/or concentration is changed towards that seen in healthy        persons;    -   (h) a compound capable of marking (e.g. with ubiquinone) at        least one of said proteins which protein is expressed at too        high relative levels in the disease for more rapid turnover or        degradation; and    -   (i) a compound capable of marking at least one of said proteins        which protein is expressed at too low relative levels for slower        turnover or degradation;        to said human. The treatment should preferably have the effect        of returning the level of the marker proteins back to the levels        seen in healthy controls.

In a presently preferred embodiment, the phosphorylation of ATP synthasebeta subunit is regulated, e.g. up-regulated, as the phosphorylation ofATP synthase beta subunit is believed to alter the function of theenzyme. The phosphorylation can be regulated by regulating the kinasesor phosphatases which phosphorylate or dephosphorylate the ATP syntasebeta subunit.

Expression of ATP synthase beta subunit is regulated by atranscriptional “co-activator of PPARγ” called PPARγ co-activator-1(PGC-1). This activator can thus be used as a means for up-regulatingATP synthase beta subunit.

In another embodiment, the invention relates to a method for diagnosingor determining the predisposition of at least one disease relating todiabetes, preferably type 2 diabetes, in a human, the method comprisesdetermining the presence, activity, concentration and/or level ofexpression of at least one marker protein in a biological sample fromthe human, wherein the marker protein is selected from the groupconsisting of:

-   -   (a) ATP synthase beta subunit or phosphorylated ATP synthase        beta subunit isoforms;    -   (b) phosphoglucomutase 1;    -   (c) heat shock protein 90 beta;    -   (d) creatine kinase B subunit;    -   (e) myosin regulatory light chain 2;    -   (f) collagen alpha1 (VI) chain;    -   (g) 78 kDa glucose-regulated protein;    -   (h) a marker protein defined by the characteristics disclosed in        table 3 (match nos: 199, 303, 391, or 511);    -   (i) an analogue having preferably at least 70% homology with any        one of the polypeptides in (a), (b), (c), (d), (e), (f), (g)        or (h) or a part thereof; and    -   (j) a derivative, precursor, analogue or modification of an        amino acid sequence in (a) to (i); and        optionally comparing the presence, activity, concentration        and/or level of expression of said protein with the presence,        activity, concentration and/or level of expression of said        protein in biological sample from at least one normal human        (i.e. not suffering from the disease in question).

The method preferably comprises:

-   -   (A) determining the increased expression, modification, activity        and/or concentration, in a biological sample from the human, of        at least one marker protein selected from the group consisting        of:        -   (a) phosphoglucomutase 1;        -   (b) heat shock protein 90 beta;        -   (c) myosin regulatory light chain 2 up-regulated isoform;        -   (d) collagen alpha1 (VI) chain;        -   (e) 78 kDa glucose-regulated protein;        -   (f) a marker protein defined by the characteristics            disclosed in table 3 (match nos: 303, or 511);        -   (g) an analogue having preferably at least 70% homology with            any one of the polypeptides in (a), (b), (c), (d), (e)            or (f) or a part thereof; and        -   (h) a derivative, analogue or modification of an amino acid            sequence in (a) to (f); and/or    -   (B) determining the decreased expression, modification, activity        and/or concentration of at least one marker protein (a        down-regulated marker protein) in a biological sample from the        human, said marker protein selected from the group consisting        of:        -   (a) ATP synthase beta subunit or phosphorylated isoforms            thereof;        -   (b) creatine kinase B subunit;        -   (c) myosin regulatory light chain 2 down-regulated isoform;        -   (d) a marker protein defined by the characteristics            disclosed in table 3 (match nos: 199, or 391);        -   (e) an analogue having preferably at least 70% homology with            any one of the polypeptides in (a), (b), (c) or (d) or a            part thereof;        -   (f) a derivative, analogue or modification of an amino acid            sequence in (a), (b), (c) or (d);            where the increased or decreased expression, modification,            activity and/or concentration is relative to a value            obtainable from a biological sample from a normal human. The            biological sample can be selected from the group consisting            of urine, blood, lymphatic fluids, saliva and tissue,            preferably muscular tissue, e.g. from the vastus lateralis            muscle. As the determination of whether a protein is            up-regulated or down-regulated serves as useful indicators            of susceptibility to a disease related to diabetes, the            invention also relates to a method for diagnosing or            determining the predisposition of at least one disease            relating to diabetes, preferably type 2 diabetes, in a            human, the method comprising determining the presence,            activity, modification, concentration and/or level of            expression of at least one marker protein (e.g. 1, 2, 3, 4,            5, 6, 7, 8, 9, 10 or more) which is present in a            significantly lower or significantly higher amount in a            sample from a person having a diabetes related disease than            in a sample from a normal person. It should be understood            that such marker proteins can be chosen from marker proteins            identified by their peptide fragment and/or gel location            (i.e. where no association to a particular gene has yet been            made—possibly because the gene has not been identified). A            person skilled in the art will be able to identify other            marker proteins, using the methods disclosed in the            examples.

In another embodiment, the invention relates to a method of determiningthe likelihood of an agent having a therapeutic effect in the treatmentof a disease related to diabetes comprising determining the level ofexpression of one or more polypeptides according to the invention beforeand after exposing a test model to said agent and comparing said levels.

Further, the invention relates to a method of determining the effect ofa compound in the treatment of a diabetes-related disease comprisingdetermining the level of expression of one or more polypeptides (e.g. 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more) according to the invention.Especially, such a method can be used to evaluate the effect of acompound used in the treatment of a diabetes-related disease comprisingdetermining the level of expression of one or more marker proteinsbefore and after exposing a test model to said agent.

A presently preferred method for determining the effect of a substancein treatment of a diabetes-related disease, is a method comprising usinga mammal or part of this mammal, which has been established to be anindividual having a high likelihood of having the disease or apredisposition (e.g. genetic) for having the disease, e.g. by use of amethod according to the invention, the method comprising administeringthe substance to the mammal or part thereof and determining the effectof the substance, preferably by determining the level of one or moremarker proteins according to the invention, before and afteradministering the substance to said mammal or part thereof.

The invention further relates to a method of determining the nature orcause of a diabetes-related disease in a human having or susceptible tosaid disease comprising establishing the level of expression of a markerprotein according to the invention in relation to a model.

In another embodiment, the invention relates to a nucleic acid fragmentwhich comprises:

-   -   (A) a nucleotide sequence which codes for a polypeptide or its        regulatory elements, which polypeptide comprises at least one        amino acid sequence selected from the group consisting of:        -   (a) ATP synthase beta subunit or phosphorylated ATP synthase            beta subunit isoforms;        -   (b) phosphoglucomutase 1;        -   (c) heat shock protein 90 beta;        -   (d) creatine kinase B subunit;        -   (e) myosin regulatory light chain 2;        -   (f) collagen alpha1 (VI) chain;        -   (g) 78 kDa glucose-regulated protein;        -   (h) a marker protein defined by the characteristics            disclosed in table 3 (match nos: 199, 303, 391, or 511);        -   (i) an analogue having preferably at least 70% homology with            any one of the polypeptides in (a), (b), (c), (d), (e),            (f), (g) or (h) or a part thereof; and        -   (j) a derivative, precursor, analogue or modification of an            amino acid sequence in (a) to (i); or    -   (B) a nucleotide sequence which hybridizes with a nucleotide        sequence according to (A), with a regulatory sequence to said        nucleotide sequence or with the complementary strand to said        sequence.

Such a nucleic acid fragment can be used for detecting the presence orlevel of a marker protein associated with a diabetes related disease;for detection of a nucleic acid sequence encoding said marker protein,e.g. as a probe or a primer for PCR, or as medication for regulation theexpression or level of a marker protein, e.g. in antisense therapy orgene therapy. Also, such a sequence can be incorporated into a vectorand used for producing the polypeptide by methods known to a personskilled in the art. Of course, the proteins can be produced by othermethods, e.g. by solid state synthesis or by purification from naturalsources, e.g. tissue.

In a still further embodiment, the invention relates to an antibody ableto bind to a marker protein (or its modification product) according tothe invention. The antibody can be polyclonal or monoclonal. Methods ofobtaining such antibodies are known to a person skilled in the art. Anantibody according to the invention can be used for detecting thepresence or level of a marker protein associated with a diabetes-relateddisease, or it can be used as a therapeutic.

In a further embodiment, the invention relates to a test kit fordiagnosing a diabetes-related disease or a predisposition, e.g. genetic,for said disease in a mammal, said test kit comprising:

-   -   (A) at least one binding means which specifically binds to at        least one marker protein according to the invention, e.g. i) an        antibody for said marker protein; ii) a nucleic acid fragment        capable of binding to said marker protein; and/or iii) a        compound (e.g. an antimimetic) capable of binding to said marker        protein;    -   (B) at least one means for detecting binding, if any, or the        level of binding, of a binding means to at least one of said        marker proteins; and, if necessary,    -   (C) at least one means for correlating whether binding, if any,        or the level of binding, to said binding means is indicative of        the individual mammal having a significantly higher likelihood        of having the disease or a (genetic) predisposition for having        the disease.

Also, the invention relates to a method for determining the effect of asubstance in treatment of a diabetes-related disease, the methodcomprising using a mammal or part of this mammal, which has beenestablished to be an individual having a high likelihood of having thedisease or a predisposition (e.g. genetic) for having the disease (e.g.by use of the method of claim 1) the method comprising administering thesubstance to the mammal or part thereof and determining the effect ofthe substance, preferably by determining the level of one or more (e.g.1, 2, 3, 4, 5, 6 or more) marker proteins according to the invention,before and after administering the substance to said mammal or partthereof.

It should be noted that the detection of any combination of more thanone of the markers would be expected to make the analysis an even morereliable indicator for the disease related to diabetes. Thus, a methodfor diagnosing or determining the predisposition of at least one diseaserelated to diabetes comprising determining the presence, activity,concentration and/or level of expression of a combination of two markerswould be preferred and three or more markers (e.g. 4, 5, 6 or moremarkers) would be strongly preferred. It is analogously suggested thattreatment with more than one compound (e.g. 2, 3, 4, 5, 6 or morecompounds) according to the invention (e.g. more than one compoundchosen from the group consisting of: a polypeptide, a nucleic acidfragment or an antibody according to the invention), said compoundscombined being able to affect the level of more than one marker protein,would make the treatment of the disease even more efficient.

By the term “diabetes related disease” is understood any disease relatedto diabetes in its broadest sense, including complications to diabetesand components of the metabolic syndrome (prior to the onset ofdiabetes). Examples of complications related to type 2 diabetes mellitusare retinopathy, neuropathy, nephropathy, macroangiopathy, insulinresistance, hyperlipidemia, hypertension, and obesity. Examples of themetabolic syndrome are obesity, dyslipidemia, glucose intolerancehypertension and other cardiovascular diseases.

The term “polypeptide” in the present invention has its usual meaning.That is an amino acid chain of any length, including a full-lengthprotein, oligopeptides, short peptides and fragments thereof, whereinthe amino acid residues are linked by covalent peptide bonds. The terms“polypeptide”, “peptide”, “amino acid sequence” and “protein” are usedinterchangeably.

The polypeptide may be chemically or biochemically modified by beingphosphorylated, methylated, sulphylated, glycosylated or by any otherforms of modification, or by the addition of any form of lipid or fattyacid, ubiquitin or any other large side groups or by containingadditional amino acids such as a signal peptide. Furthermore, thepolypeptide may be cleaved e.g. by processing at its N- or C-termini orbe spliced to remove an internal sequence.

Each polypeptide may thus be characterized by specific amino acids andbe encoded by specific nucleic acid sequences. It will be understoodthat such sequences include analogues and variants produced byrecombinant or synthetic methods wherein such polypeptide sequences havebeen modified by substitution, insertion, addition or deletion of one ormore amino acid residues in the recombinant polypeptide and still beingimmunogenic in any of the biological assays described herein.Substitutions are preferably “conservative”. Conservative substitutionsare known to a person skilled in the art. Preferably, amino acidsbelonging to the same grouping (non-polar (G, A, P, I, L and V),polar-uncharged (C, S, T, M, N and Q), polar-charged (D, E, K and R) andaromatic (H, F, W and Y)). Within these groups, amino acids may besubstituted for each other, but other substitutions are of coursepossible.

Each polypeptide is encoded by a specific nucleic acid sequence. It willbe understood that such sequences include analogues and variants hereofwherein such nucleic acid sequences have been modified by substitution,insertion, addition or deletion of one or more nucleic acid residues(including the insertion of one or more introns (small or large)).Substitutions are preferably silent substitutions in the codon usage,which will not lead to any change in the amino acid sequence, but may beintroduced to enhance the expression of the protein.

In the present context the term “substantially pure polypeptidefragment” means a polypeptide preparation which contains at most 5% byweight of other polypeptide material with which it is nativelyassociated (lower percentages of other polypeptide material arepreferred, e.g. at most 4%, at most 3%, at most 2%, at most 1%, and atmost ½%). It is preferred that the substantially pure polypeptide is atleast 96% pure, i.e. that the polypeptide constitutes at least 96% byweight of total polypeptide material present in the preparation, andhigher percentages are preferred, such as at least 97%, at least 98%, atleast 99%, at least 99.25%, at least 99.5%, and at least 99.75%. It isespecially preferred that the polypeptide fragment is in “essentiallypure form”, i.e. that the polypeptide fragment is essentially free ofany other protein with which it is natively associated, i.e. free of anyother protein from a mammal. This can be accomplished by preparing thepolypeptide of the invention by means of recombinant methods in a hostcell as known to a person skilled in the art, or by synthesizing thepolypeptide fragment by the well-known methods of solid or liquid phasepeptide synthesis, e.g. by the method described by Merrifield(Merrifield, R. B. Fed. Proc. Am. Soc. Ex. Biol. 21: 412, 1962 and J.Am. Chem. Soc. 85: 2149, 1963) or variations thereof, or by means ofrecovery from electrophoretic gels.

The invention also encompasses isoforms, derivatives, precursors,truncates (such as mature forms), analogues and mimetics of the abovementioned polypeptides. Such an isoform, derivative, analogue andmimetic preferably have the same activity, e.g. the same kind ofenzymatic activity, as the polypeptide from which it is derived. Theisoform, derivative, analogue or mimetic can have a lower level ofactivity, the same level or preferably, a higher level of activity thanthe parent polypeptide.

The term “isoform” refers to a family of related proteins (i.e. multipleforms of the same protein) that differ somewhat in their amino acidsequence. They can be produced by different genes or by alternativesplicing of RNA transcripts from the same gene. Thus, the term “isoform”comprises homologous sequences of amino acid residues interspersed withvariable sequences. Also, the term “isoform” comprises a form of theprotein which has been post translationally processed, e.g.phosphorylated (phospho-isoform).

A “peptide mimetic” is a molecule that mimics the biological activity ofa peptide but is no longer peptidic in chemical nature. By strictdefinition, a peptidomimetic is a molecule that no longer contains anypeptide bonds (that is, amide bonds between amino acids). However, theterm peptide mimetic is sometimes used to describe molecules that are nolonger completely peptidic in nature, such as pseudo-peptides,semi-peptides and peptoids. Whether completely or partially non-peptide,peptidomimetics according to this invention provide a spatialarrangement of reactive chemical moieties that closely resembles thethree-dimensional arrangement of active groups in the peptide on whichthe peptidomimetic is based. As a result of this similar active-sitegeometry, the peptidomimetic has effects on biological systems, whichare similar to the biological activity of the peptide. The presentinvention encompasses peptidomimetic compositions which are analogs thatmimic the activity of biologically active peptides according to theinvention, i.e. the peptidomimetics can be used for treatment ofdiabetes related diseases. The peptidomimetic of this invention arepreferably substantially similar in both three-dimensional shape andbiological activity to the peptides or active sites of such as set forthabove.

Alternatively, the mimetic can be an ‘antimimetic’. In other words, amolecule that can fit into and block the active site of the protein, orbind to binding sites or sites of interaction with other biologicalmolecules and so interfere with the function of the protein. Mostcurrent drugs are of this type. Such antimimetics that are capable ofinteracting with the polypeptides of the invention are encompassed bythe present invention.

An aptamer is a compound that is selected in vitro to bindpreferentially to another compound (in this case the identifiedproteins). Often, aptamers are nucleic acids or peptides because randomsequences can be readily generated from nucleotides or amino acids (bothnaturally occurring or synthetically made) in large numbers but ofcourse they need not be limited to these.

There are clear advantages for using a mimetic of a given peptide ratherthan the peptide itself, because peptides commonly exhibit twoundesirable properties: (1) poor bioavailability; and (2) short durationof action. Peptide mimetics offer an obvious route around these twomajor obstacles, since the molecules concerned are small enough to beboth orally active and have a long duration of action. There are alsoconsiderable cost savings and improved patient compliance associatedwith peptide mimetics, since they can be administered orally comparedwith parenteral or transmucosal administration for peptides.Furthermore, peptide mimetics are much cheaper to produce than peptides.Finally, there are problems associated with stability, storage andimmunoreactivity for peptides that are not experienced with peptidemimetics.

Thus, the peptides described above have utility in the development ofsuch small chemical compounds with similar biological activities andtherefore with similar therapeutic utilities. The techniques ofdeveloping peptidomimetics are conventional. Thus, peptide bonds can bereplaced by non-peptide bonds that allow the peptidomimetic to adopt asimilar structure, and therefore biological activity, to the originalpeptide. Further modifications can also be made by replacing chemicalgroups of the amino acids with other chemical groups of similarstructure. The development of peptidomimetics can be aided bydetermining the tertiary structure of the original peptide by NMRspectroscopy, crystallography and/or computer-aided molecular modelling.These techniques aid in the development of novel compositions of higherpotency and/or greater bioavailability and/or greater stability than theoriginal peptide [Dean (1994), BioEssays, 16: 683-687; Cohen andShatzmiller (1993), J. Mol. Graph. 11: 166-173; Wiley and Rich (1993),Med. Res. Rev., 13: 327-384; Moore (1994), Trends Pharmacol. Sci., 15:124-129; Hruby (1993), Biopolymers, 33: 1073-1082; Bugg et al. (1993),Sci. Am., 269: 92-98, all incorporated herein by reference]. Once apotential peptidomimetic compound is identified, it may be synthesizedand assayed using the diagnostic assay described herein or anappropriate disease suppressor assay [see, Finlay et al. (1983), Cell,57: 1083-1093 and Fujiwara et al. (1993), Cancer Res., 53: 4129-4133,both incorporated herein by reference], to assess its activity.

Thus, through use of the methods described above, the present inventionprovides compounds exhibiting enhanced therapeutic activity incomparison to the polypeptides described above. The peptidomimeticcompounds obtainable by the above methods, having the biologicalactivity of the above named peptides and similar three dimensionalstructure, are encompassed by this invention. It will be readilyapparent to one skilled in the art that a peptidomimetic can begenerated from any of the modified peptides previously described or froma peptide bearing more than one of the modifications previouslydescribed. It will furthermore be apparent that the peptidomimetics ofthis invention can be further used for the development of even morepotent non-peptidic compounds, in addition to their utility astherapeutic compounds.

By the terms “nucleic acid fragment” and “nucleic acid sequence” areunderstood any nucleic acid molecule including DNA, RNA, LNA (lockednucleic acids), PNA, RNA, dsRNA and RNA-DNA-hybrids. Also included arenucleic acid molecules comprising non-naturally occurring nucleosides.The term includes nucleic acid molecules of any length, e.g. from 10 to10000 nucleotides, depending on the use. When the nucleic acid moleculeis for use as a pharmaceutical, e.g. in DNA therapy, or for use in amethod for producing a polypeptide according to the invention, amolecule encoding at least a part of the polypeptide is preferably used,having a length from about 18 to about 1000 nucleotides, the moleculebeing optionally inserted into a vector. When the nucleic acid moleculeis used as a probe, as a primer or in antisense therapy, a moleculehaving a length of 10-100 is preferably used. According to thisinvention, other molecule lengths can be used, for instance a moleculehaving at least 12, 15, 21, 24, 27, 30, 33, 36, 39, 42, 50, 60, 70, 80,90, 100, 200, 300, 400, 500 or 1000 nucleotides (or nucleotidederivatives), or a molecule having at most 10000, 5000, 4000, 3000,2000, 1000, 700, 500, 400, 300, 200, 100, 50, 40, 30 or 20 nucleotides(or nucleotide derivatives). It should be understood that these numberscan be freely combined to produce ranges.

The term “stringent” when used in conjunction with hybridizationconditions is as defined in the art, i.e. the hybridization is performedat a temperature not more than 15-20° C. under the melting point (Tm) ofthe nucleic acid fragment, cf. Sambrook et al Molecular Cloning; Alaboratory manual, Cold Spring Harbor Laboratories, NY, 1989, pages11.45-11.49. Preferably, the conditions are “highly stringent”, i.e.5-10° C. under the melting point (Tm).

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations thereof such as “comprises” or“comprising”, will be understood to imply the inclusion of a statedelement or integer or group of elements or integers but not theexclusion of any other element or integer or group of elements orintegers.

The term “sequence identity” indicates a quantitative measure of thedegree of homology between two amino acid sequences of equal length orbetween two nucleotide sequences of equal length. If the two sequencesto be compared are not equal length, they must be aligned to bestpossible fit with the insertion of gaps or alternatively truncation atthe end of the protein sequences. The sequence identity can becalculated as

$\frac{\left( {N_{ref} \cdot N_{dif}} \right)100}{N_{ref}},$wherein N_(dif) is the total number of non-identical residues in the twosequences when aligned and wherein N_(ref) is the number of residues inone of the sequences. Hence, the DNA sequence AGTCAGTC will have asequence identity of 75% of the sequence AATCAATC (N_(dif)=2 andN_(ref)=8). Sequence identity can alternatively be calculate by theblast program e.g. the BLASTP program (Pearson W. R and D. J. Lipman(1988) PNAS USA 85:2444-2448. In one aspect of the invention, alignmentis performed with the sequence alignment method Clustalw with defaultparameters as described by Thompson J., et al Nucleic Acids Res 1994 22:4673-4680.

A preferred minimum percentage of sequence identity is at least 70%,such as at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, and at least 99.5%.

The invention also relates to the use of a polypeptide or nucleic acidof the invention for use as therapeutic vaccines as have been describedin the literature exemplified by Lowry, D. B. et al 1999, Nature 400:269-71.

A monoclonal or polyclonal antibody, which is specifically reacting witha polypeptide of the invention in an immuno assay, or a specific bindingfragment of said antibody, is also part of the invention. The antibodiescan be produced by methods known to a person skilled in the art. Thepolyclonal antibodies can be raised in a mammal, for example, by one ormore injections of a polypeptide according to the present invention and,if desired, an adjuvant. The monoclonal antibodies according to thepresent invention may, for example, be produced by the hybridoma methodfirst described by Kohler and Milstein, Nature, 256:495 (1975), or maybe produced by recombinant DNA methods such as described in U.S. Pat.No. 4,816,567. The monoclonal antibodies may also be isolated from phagelibraries generated using the techniques described by McCafferty et al,Nature, 348:552-554 (1990), for example. Methods for producingantibodies are described in the literature, e.g. in U.S. Pat. No.6,136,958.

In diagnostics, an antibody, a nucleic acid fragment and/or apolypeptide of the invention can be used either alone, or as aconstituent in a composition. Such compositions are known in the art,and comprise compositions in which the antibody, the nucleic acidfragment or the polypeptide of the invention is coupled, preferablycovalently, to at least one other molecule, e.g. a label (e.g.radioactive or fluorescent) or a carrier molecule.

The present invention is further directed to methods for using thecompounds described above to therapeutically and/or prophylacticallytreat a patient for a diabetes related disease.

The methods of the present invention include the steps of: a)incorporating one or more of the compounds of the present invention in asuitable pharmaceutical carrier; and b) administering either atherapeutically effective dosage or a prophylactically effective dosageof the compound or compounds incorporated in the carrier to a patient.

The term “suitable pharmaceutical carrier” refers to any carrier knownin the pharmaceutical arts for administration of compounds to a patient.Any suitable pharmaceutical carrier can be used according to the presentinvention, so long as compatibility problems do not arise.

Administration of an effective dosage to a patient can be accomplishedby parenteral injection, such as intravenously, intrathecally,intramuscularly or intra-arterially. The compounds can also beadministered orally or transdermally, or by any other means known tothose skilled in the art, e.g. by means of an inhalator or a nasalspray. Oral administration is presently preferred.

As used herein, the term “therapeutically effective amount” refers tothat amount of one or more of the compounds of the present inventionrequired to therapeutically treating a patient. Such treatment isappropriate for subjects having a diagnosed diabetes related disease.Similarly, the term “prophylactically effective amount” refers to thatamount of one or more of the compounds of the present invention neededto prophylactically treat a patient. Such treatment is appropriate forsubjects who, for example, have not yet established any clinicalsymptoms of a diabetes related disease. It could be advantageous tostart a prophylactic treatment as soon it is determined that the subjectis in risk for developing a diabetes related disease, e.g. by means ofdetermination of a predisposition for diabetes by having an alteredlevel of markers.

As will be appreciated by a person skilled in the art, the dosage of acompound given, the route of administration and the duration of therapywill be dependent on not only the type of compound and its effectivenessin treating the disease but also upon the individual being treated,taking into consideration such factors as the body weight of thepatient, other therapies being employed to treat the patient, and thecondition, clinical response and tolerance of the patient. Dosage,administration, and duration of therapy can be determined by one skilledin the art upon evaluation of these and other relevant factors.

Determination of the levels of the found markers of type 2 diabetes on amuscle biopsy specimen from a subject genetically prone to develop type2 diabetes can be used to determine the need for intervention in orderto prevent type 2 diabetes or to treat the prediabetic state in the formof medical treatment, guidelines for diet, physical activity etc. If forexample the levels of the down-regulated phospho-isoform of ATP synthasebeta subunit described above is less than 0.5 % of the totalprotein-related IOD on a 2D-gel image (from an IPG gel covering the pHrange from 4 to 7 prepared as described herein), the probability ofhaving type 2 diabetes or a prediabetic state is greater than 85%. Inthe same way, if the levels of creatine kinase B is less than 0.25 %IODon a 2D-gel image, the probability of having type 2 diabetes or aprediabetic state is greater than 80%. And if the levels of both markersof type 2 diabetes are lower than these levels the probability of havingtype 2 diabetes or a prediabetic state is even greater.

Substances (hormones, kinases, phosphatases, small molecules etc.) thatcan modulate the phosphorylation and/or expression of ATP synthasebeta-subunit are under investigation. Substances that modulate thephosphorylation and/or expression of ATP synthase beta-subunit in amanner that results in improvements of the physiological parametersrelated to type 2 diabetes such as insulin sensitivity, glucose uptakeand blood glucose can be used directly, whereas substances with theopposite effect can be used for the development of inhibitors. Ourstudies suggest that therapeutic activation of the kinase, whichphosphorylates ATP synthase beta-subunit, will result in improved ATPsynthesis, with subsequent improvements in insulin sensitivity, glucoseuptake and blood glucose.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1: Protein markers of type 2-diabetes in skeletal muscleRepresentative 2D gel image of human skeletal muscle from a diabeticsubject. Protein spots were separated in the first dimension by IPG gels(pI 4-7) and visualised by silver staining. The numbers correspond tothe protein spots that were significantly up-regulated (underlined) ordown-regulated in muscle of type 2-diabetic subjects. Of these fifteenproteins, eleven were identified by MALDI-MS (Table 2).

FIG. 2: Phosphorylation of ATP synthase β-subunit (ATPsyn-β) in humanskeletal muscle

FIG. 2A: Comparison of enlarged regions of a silver-stained 2D gel ofskeletal muscle and a [³²P]-labelled 2D gel of cultured human skeletalmuscle cells (myoblasts) showing that all four isoforms arephosphorylated isoforms of ATPsyn-β.

FIG. 2B: The large spot located to the right of ATPsyn-β (spot 180) onthe silver-stained gel was also identified as an ATPsyn-β isoform byMALDI-TOF MS analysis. The MALDI peptide mass map of this ATPsyn-βisoform showed the presence of a phosphorylated peptide (two peaks witha mass difference of 80 Da) with the most probable phosphorylation sitebeing threonine at the N-terminal end of the peptide.

FIG. 2C: The sequence coverage of this more abundant ATPsyn-β isoformwas 59% and 29 out of 45 measured peptides were matched to this proteinas indicated (bold). The potential phosphorylation site of thephosphorylated peptide (marked by a box) was Thr213 within thenucleotide-binding region (underlined).

FIG. 3: Potential roles for the observed protein markers of type2-diabetes in skeletal muscle metabolism

FIG. 3A-3B: Correlation between expression of the down-regulated ATPsynthase β-subunit (ATPsyn-β) isoform and fasting plasma glucose levelsin DM2 subjects (r=−0.75; P=0.020), and fasting plasma FFA levels incontrol subjects (r=−0.81; P=0.049).

FIG. 3C-3D: Correlation between expression of creatine kinase B andfasting plasma glucose levels (r=−0.82; P=0.007) and expression of thedown-regulated ATPsyn-β isoform (r=0.71; P=0.033) in DM2 subjects.

FIG. 3E: Proposed scheme for the putative roles of the protein markersof type 2 diabetes (marked with grey) in skeletal muscle metabolism. TheF₁ portion of ATP synthase contains the three catalytic β-subunits, andcomplex IV of the respiratory chain is identical to cytochrome Coxidase. ROS, reactive oxygen species; PKB, protein kinase B; eNOS,endothelial nitric oxide synthase; AMPK, AMP-activated protein kinase;TCA cycle, tricarboxylic acid cycle; mtCK, mitochondrial creatinekinase; GLUT4, glucose transporter 4; GS, glycogen synthase; IR, insulinreceptor; G-6-P, glucose-6-phosphate and G-1-P, glucose-1-phosphate.Remaining abbreviations are explained in the text section.

FIG. 4: Two different MRLC2 isoforms differently expressed in diabeticmuscle Spot A was down-regulated, whereas spot I was up-regulated in DM2subjects. Spots A, B and C were all identified by MALDI-MS as the MRLC2ventricular/cardiac muscle isoform (database acc. no: P10916) andprobably represent different phospho-isoforms. Spots I, II and III wereall identified as another MRLC2 isoform (database acc. no: AAK52797),and they probably also represent different phospho-isoforms. There isonly 72% homology between the two different isoforms of MRLC2.

EXPERIMENTIAL

Proteome analysis offers the possibility to study thousands of proteinsas well as their post-translational modifications simultaneously, and isa promising technique in the study of complex diseases such as type 2diabetes (5). The high-resolution proteome technology effectivelyseparates and identifies proteins with high success rate. Compared toanalyses of mRNA expression, proteome analysis offer the possibility ofrelative quantification of changes in protein expression as well asidentification and quantitation of post-translatory protein modificationsuch as phosphorylation, methylation and cleavage. Post-translationalmodifications are often required for the functional activation of aprotein and hence, may be of pathogenetic importance.

The present inventors have used proteome analysis to select and identifyproteins associated to diabetes. These proteins, in themselves, eitherup-regulated or down-regulated, are indicators of diabetes in a patient.The pattern of regulation of a grouping of these proteins also serves asan indicator of diabetes. These proteins can be used as targets for thetreatment of diabetes or they can be used as therapeuticals fortreatment of the disease.

Thus, the determination of whether a protein is up-regulated ordown-regulated serves as useful indicators of diabetes susceptibility.The pattern of up and down regulation may also serve as an indicator.That is to say that the level of expression of more than one protein isestablished and the pattern of expression of a grouping of proteins isused as an indicator. Obviously, the reliability of identificationincreases as the number in the group increases.

Muscle Samples

Muscle samples were collected from 10 type 2 diabetic (diabetes group)and 10 healthy subjects (control group). Subjects with type 2 diabetes(fasting C-peptide>600 pmol/l and GAD65 antibody negative) were treatedby diet only or in combination with oral antidiabetics, which werewithdrawn 2 weeks before the study. Subjects were instructed to refrainfrom excessive physical exercise for 48 h and to fast for 10 h(overnight) before the study. Normal glucose tolerance was confirmed innon-diabetic subjects and fasting concentrations of plasma glucose andFFA and serum insulin and C-peptide were assayed as described previously(26). Percutaneous needle biopsy of the vastus lateralis muscle wasperformed with a biopsy pistol, and the muscle specimens (˜25 mg) wereimmediately blotted free of blood, fat and connective tissue and frozenin liquid nitrogen. The study protocol was performed in accordance withthe Helsinki Declaration.

Sample Preparation

The frozen muscle samples were homogenized for 25 min in 100 μl oficecold DNase/RNase buffer (20mmol/l Tris-HCl buffer pH 7.5 containing30 mmol/l NaCl, 5 mmol/l CaCl_(2,) 5 mmol/l MgCl₂ and 25 μg/ml RNaseA/DNase I (Worthington, Freehold, N.J.)). After homogenization, thesamples were lyophilized overnight and then dissolved in 120 μl of lysisbuffer (7 M urea (ICN Biomedicals), 2 M thiourea (Fluka), 2% CHAPS, 0.4%DTT (Sigma), 0.5% Pharmalyte 3-10 and 0.5% Pharmalyte 6-11 (AmershamPharmacia Biotech, Sweden)) under continuous shaking.

Protein and CPM Determination

The protein concentration in the samples was determined using theBradford method, which was adopted for use with lysis buffer asdescribed before (29).

Two Dimensional Electrophoresis

First dimension gel electrophoresis was performed on IPG 4-7 gradientgels (Amersham Pharmacia Biotech). Rehydration buffer for IPG4-7 stripswas identical with lysis buffer used for sample preparation and thesample was applied by in-gel rehydration. 400 μg of protein was loadedon each gel. Focusing was performed on Multiphor II at 20° C. usingvoltage/time profile linearly increasing from 0 V to 600 V for 2:15 h,from 600 V to 3500 V for 1 h and 3500 V for 13:30 h. After focusing,strips were equilibrated two times 15 min in equilibration buffer (6Murea, 2% SDS, 30% Glycerol, 50 mM Tris-HCL pH 8.8, 1% DTT). The gelswere kept frozen in −80° C. between the equilibration steps. SDS PAGEsecond dimension was performed using Protean II Multi Cell 2-DElectrophoresis System (Bio-Rad) and laboratory-made single percentagegels (12.5% acrylamide; acrylamide: N,N′-ethylene-bis-acrylamide ratio200:1). The gels were run overnight at 20° C. at constant current.Running buffer was recirculated to maintain pH, temperature and saltconcentrations.

Protein Visualization and Computer Analysis

After the second dimension, proteins were visualized usingsilver-staining method as described (29). All gel images were analyzedby the same person using Bio Image computer program (version 6.1, B. I.System Corporation). The expression of each protein was measured andexpressed as its percentage integrated optical density (%IOD) (apercentage of the sum of all the pixel grey level values within boundaryof the spot in question compared to that of all detected spots). Forcomparison, the present inventors have used 6 gels from the controlgroup and 9 from the diabetes group (the remaining gels revealed proteindegradation that occurred during the sample preparation procedure andthus could not be used). Images from each group were matched andcompared. The average value of spot %IOD and standard deviation for eachprotein in each group were calculated and then compared using Student'st-test. Protein spots whose expression was found different at thesignificance level of 95% were selected for further analysis.

Mass Spectrometric Protein Identification

Proteins of interest were cut out from gels and after in-gel digestionanalysed by MALDI mass spectrometry (27, 28, 29). The obtained massspectra were internally calibrated using trypsin autodigestion peptidesand the masses were used to search NOBI database using the ProFound andfurther analysed with FindPept and FindMod programs(www.proteometrics.com). Database searches were performed using thefollowing attributes with minor modification needed for each program:all species, no restrictions for molecular weight and protein pl,trypsin digest, one missed cleavage allowed, cysteines modified byacrylamide, and oxidation of methionines possible, mass tolerancebetween 0.1-0.5 Da. Identification was considered positive, when atleast 5 peptides matched the protein with no sequence overlap.

[³²P]-Labelling of Human Myoblasts

Human skeletal muscle cell cultures were established as previouslydescribed (Hemmer et al, 30). Myoblasts were grown in 12-wells plates,and growing cell medium was changed to DMEM containing 5 mM glucose andsupplemented with 10% foetal calf serum 1 day before the labellingexperiment. Prior to labelling, cells were incubated in serum-free DMEMmedium containing 0.2 % bovine serum albumin (BSA) for 2.5 hours.Phosphate groups in proteins of human myoblasts were biosyntheticallylabelled by incubating the myoblasts in 300 μl serum-free phosphate-freeDMEM medium (ICN, Ohio) supplemented with 2 mM L-glutamine (LifeTechnologies, Paisley, Scotland), 0.2% BSA and 300 μCi[³²P]-orthophosphate (Amersham Biosciences) for 2.5 h. Immediatelyafter, labelling medium was removed and cells were lysed in 400 μl lysisbuffer as described above. Determination of [³²P]-orthophosphateincorporation into myoblast proteins was performed using TCA(trichloroacetic acid) precipitation as described (29). Two-dimensionalelectrophoresis was run as described above loading a cell lysate volumecorresponding to 4×10⁵ cpm on the gel. [³²P]-labelled proteins ofmyoblasts were visualised by exposing dried gels to phosphoimager plates(AGFA).

Results

The present inventors collected biopsies of the vastus lateralis musclefrom age and gender matched control and type 2 diabetic (DM2) subjectsto compare the in vivo protein expression profile of resting skeletalmuscle in the postabsorptive state by proteome analysis (Table 1). Thepresent inventors separated muscle proteins by two-dimensional (2D) gelelectrophoresis and proteins were visualized by silver-staining method.The present inventors were able to match and quantitate 489 spots ineach gel image using computerized image analysis. Fifteen protein spotswere expressed at statistically different levels in the two groups (FIG.1). These protein markers for DM2 were excised from the 2D gels, andfollowing in gel tryptic digestion, submitted to matrix assisted laserdesorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS)and database searching for identification. Eleven of them have beenidentified; three metabolic enzymes, two chaperone proteins, andisoforms of two structural proteins (Table 2).

Phoshoglucomutase 1 (PGM-1) was significantly up-regulated in DM2subjects (table 2). PGM1 is a glycolytic enzyme, that plays a pivotalrole in glycogen metabolism.

The present inventors found that expression of heat-shock protein 90beta (HSP90β) was significantly up-regulated in muscle from DM2 subjects(Table 2). The type of stress that increases expression of HSP90β indiabetic muscle is currently unknown.

One isoform of myosin regulatory light chain 2 (MRLC2) was significantlydown-regulated while another isoform of MRLC2 was significantlyup-regulated in DM2 subjects (Table 2). The function of MRLC2 inskeletal muscle is only partially understood. The potential implicationsof these changes in expression of MRLC2 isoforms are therefore unclear.

The down-regulated isoform of MRLC2 is identified as theventricular/cardiac muscle isoform of MRLC2 (database accession numberP10916), and the up-regulated isoform is identified as another isoformhaving the database accession number AAK52797. There is only 72%homology between the two isoforms.

Creatine kinase B (CK-B) was significantly down-regulated in DM2subjects (Table 2). The present inventors found a negative correlationbetween plasma glucose and CK-B levels (FIG. 3C) in diabetic musclesuggesting a role for CK-B in glycolysis during hyperglycemia. Inaddition, the present inventors found a positive correlation of CK-Bwith the down-regulated ATPsyn-β phosphoisoform (FIG. 3D) in DM2subjects.

The spot identified as ATP synthase beta subunit (ATPsyn-β) wassignificantly down-regulated in DM2 subjects (Table 2), and appearedwithin a series of four spots with identical molecular weights butdifferent pI values, indicating heterogenous charge variants of the sameprotein (FIG. 2A). To further characterize the modification of ATPsynthase β-subunit, we carried out 2-D gel electrophoresis of[³²P]-labelled human skeletal muscle cells (myoblasts). These 2-D gelsrevealed that all of the four identified ATPsynβ isoforms are in factphosphorylated isoforms (FIG. 2A), and that a putative non-modifiedvariant was below the level of detection by silver staining. MALDI-MSanalysis and database searching for phosphopeptides from the trypticdigest of the three most basic phospho-isoforms of ATPsynβ, includingthe down-regulated ATPsynβ spot (no. 180), clearly demonstrated thepresence of a phosphorylated residue most likely at position Thr213 inthe nucleotide-binding domain of ATPsynβ (FIG. 2B-C). Tyrosinesulphation may give rise to the same pattern on 2-D gels and the sameincrease in peptide mass (80 Da) as phosphorylation. However, using theconsensus features of a tyrosine sulphation site (31, 32), such siteswere excluded in the sequence of ATPsynβ.

The [³²P]-labelling of all 4 ATPsynβ isoforms and the mass spectrometrydata therefore indicate that ATPsynβ is regulated by multisitephosphorylation, and the present data demonstrate that the catalyticβ-subunit of human F1-ATP synthase is regulated by phosphorylation inskeletal muscle, and that this regulation might be altered in DM2.

Also, 78-kDA glucose-regulated protein (GRP78) was significantlyup-regulated in muscle tissue of patients with type 2 diabetes. Weobserved increased levels of GRP78 in type 2 diabetic subjects, and ofthe three isoforms identified only the most basic (non-phosphorylated)and most active isoform was significantly up-regulated.

Four spots, each presumably representing an isoform of alpha-1 chain oftype VI collagen (α1(VI) collagen), demonstrated that this protein andisoforms thereof were significantly up-regulated in patients with type 2diabetes.

In control subjects the present inventors observed a negativecorrelation between the down-regulated expression levels of the ATPsyn-βphospho-isoform and fasting plasma FFA (FIG. 3B), whereas in DM2subjects the expression of this phospho-isoform did not correlate withFFA but instead correlated inversely with fasting plasma glucose (FIG.3A). This is interesting, because a positive correlation of uncouplingprotein 3 mRNA levels in muscle with circulating levels of FFA innon-diabetic subjects in the fasting state (23) seems to be absent inDM2 subjects (24) and replaced by an association to hyperglycemia (25),exactly as observed with the down-regulated ATPsyn-β phospho-isoform.

In summary, using proteome analysis the present inventors found thatfifteen proteins surprisingly were up- or down regulated in muscle ofsubjects with DM2. Eleven of these proteins were positively identifiedby mass spectrometry. The proteins can be used as protein markers of DM2in skeletal muscle in the post absorptive state. The type VI collagenisoforms have not been disclosed as being up-regulated in skeletalmuscular tissue in diabetics, and the rest of these eleven proteins havenot previously been directly associated with DM2.

Most surprisingly, the present inventors demonstrated that the catalyticβ-subunit of F₁-ATP synthase is phosphorylated. In addition, the presentinventors found expression of a β-subunit phospho-isoform in diabeticmuscle to be reduced and to correlate inversely with plasma glucoselevels. These data show a role for phosphorylation of ATPsyn-β in theregulation of ATP synthesis, and indicate that alterations in theregulation of this protein contribute to pathogenesis of DM2.

TABLE 1 Clinical characteristics Control DM2 P n 6 9 Age (years) 46.1 ±1.5  44.8 ± 1.5  ns Body mass index (kg/m²) 25.7 ± 1.2  33.3 ± 1.9 0.01  Male/female 3/3 5/4 ns Fasting glucose (mmol/l) 5.3 ± 0.1 8.5 ±0.7 0.002 Fasting FFA (mmol/l) 0.28 ± 0.04 0.44 ± 0.08 ns Fastinginsulin (pmol/l) 37 ± 11 93 ± 12 0.005 Fasting C-peptide (pmol/l) 575 ±120 1195 ± 93  0.001 Clinical characteristics of type 2 diabetic (DM2)and control subjects. Data represent mean ± s.e.m. P values arecalculated by Student's t-test; ns, not significant. Male/female ratiowas tested by Chi-square test.

TABLE 2 Protein markers of type 2 diabetes identified by MALDI TOF MSanalysis Spot Database Theoretical Sequence Matched Control groupDiabetic group no. Protein acc. no. pI/mW Coverage peptides % IOD ± SEM% IOD ± SEM 180 ATP synthase β-subunit P06576 5.0/52 56% 19 0.56 ± 0.05 0.41 ± 0.03 ** 10 Collagen alpha 1 (VI) chain P12109  5.3/110 10% 100.06 ± 0.01 0.09 ± 0.01 * 11 Collagen alpha 1 (VI) chain P12109  5.3/110 5% 5 0.04 ± 0.00  0.06 ± 0.01 ** 407 Collagen alpha 1 (VI) chain P12109 5.3/110  6% 6 0.05 ± 0.01 0.08 ± 0.01 * 520 Collagen alpha 1 (VI) chainP12109  5.3/110  6% 5 0.06 ± 0.01 0.09 ± 0.01 * 541 Creatine kinase BP12277 5.3/43 42% 12 0.32 ± 0.04 0.20 ± 0.02 * 375 Glucose regulatedprotein 78 P11021 4.9/78 28% 12 0.06 ± 0.01 0.08 ± 0.01 * 81 Heat shockprotein 90 beta P08238 5.0/84 15% 11 0.03 ± 0.01 0.05 ± 0.00 * 295Myosin regulatory light chain 2 (A) P10916 4.9/19 58% 9 1.26 ± 0.10 0.92± 0.10 * 445 Myosin regulatory light chain 2 (B) AAK52797 4.9/19 52% 70.30 ± 0.08 0.69 ± 0.12 * 456 Phosphoglucomutase 1 P36871 6.2/62 25% 120.14 ± 0.04 0.28 ± 0.04 * Identified protein markers of type 2 diabetesin skeletal muscle are given with their database accession numbers,theoretical molecular weight (mW) and pI. The expression levels of theseprotein markers are given as mean ± SEM of the percentage integratedoptical density (% IOD) of proteins. * P < 0.05 vs. control; ** P < 0.01vs. control.

TABLE 3 Protein markers of type 2 diabetes not identified by MS MatchExpression (% IOD) No DM2 Control ~pI § ~mW (kDa) § 199 0.025 ± 0.0150.067 ± 0.032** ~5.2 ~45 303 0.154 ± 0.049 0.098 ± 0.047*  ~5.6 ~55 3910.028 ± 0.028 0.072 ± 0.046*  ~5.0 ~25 511 0.052 ± 0.020 0.023 ± 0.013**~4.9 ~65 Expression levels presented as percentage integrated opticaldensity (% IOD), mean ± SD. §, approximate values predicted fromlocation on the 2 dimensional gel. **P < 0.01; *P < 0.05. The expressionlevel % IOD is given as determined using IPG gels covering the pH rangefrom nominally 4 to 7. The actual values may be different if the gelsystem used is different to that used here.Testing for Up- or Down-Regulation of the Proteins

A small percutaneous needle biopsy is collected from the vastuslateralis muscle and expelled into a hypotonic solution (to burst thecells and mitochondria). This solution contains protease, kinase andphosphorylase inhibitors. The sample is shaken for 15 minutes to releasethe proteins and then 100 μl is applied to the appropriate wells ofappropriately prepared ELISA microtitre plates.

The microtitre plates have been coated with antibodies (eithermonoclonal or polyclonal, one in each row (except for the standardsactin and rubisco)) which specifically recognise ATPsynthase, thephosphorylated form of ATP synthase, and some or all of the otherproteins identified in table 2. Antibodies against other proteins, forexample glycogen synthase can also be included. In addition a positivecontrol is included (for example actin), and a negative control (i.e. aprotein that should not be recognised like the plant protein rubisco) isincluded to make sure that the immunological reactions are workingcorrectly. Furthermore, the plate contains standard curves for thevarious proteins in question as illustrated below. These standard curvescan be made using recombinant proteins, modified or cleavedappropriately.

The microtitre plates are then left for 1 hr at 4° C., and then they arewashed, developed and read in an ELISA plate reader. Certain readers arealso capable of calculating the actual amount of the specific protein inthe unknown sample with reference to the standard curve. The plate isrejected if actin does not stain or if rubisco does.

Each protein is known to have an average value and maximum and minimumtolerance limits (corresponding to the usual range seen in healthyindividuals). When the protein exceeds these limits, the person can beconsidered as developing a particular disease related to diabetes type2. Positive indications from 2 or more of the diagnostic markers aremuch more significant than from only one. For example, high creatinekinase B results are seen in patients that have recently experiencedischaemia and so this marker—on its own—is not sufficient to indicatediabetes type 2.

There are of course many other formats and procedures that are known toone skilled in the art which could be used to reach essentiallyidentical diagnostic conclusions. The data obtained herewith can also becombined with other data relevant to the development of diabetes, forexample serum glucose, or free fatty acid levels in order to increasethe reliability of specificity of the diagnosis or prognosis.

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1. A method for indicating type 2 diabetes in a human, the methodcomprising determining the, activity, concentration and/or level ofexpression of at least two marker proteins in a biological sample fromthe human, and comparing the, activity, concentration and/or level ofexpression of said proteins with the, activity, concentration and/orlevel of expression of said proteins in a biological sample from atleast one normal human, wherein the sample is selected from the groupconsisting of urine, blood, lymphatic fluids and skeletal muscletissues, and wherein the marker proteins are selected from the groupconsisting of: (a) ATP synthase beta subunit or phosphorylated ATPsynthase beta subunit isoforms; (b) phosphoglucomutase 1; (c) heat shockprotein 90 beta; (d) creatine kinase B subunit; (e) myosin regulatorylight chain 2; (f) collagen alphal (VI) chain; and (g) 78 kDaglucose-regulated protein.